Addition of Sugar, Amino Acids and Corn Steep Liquor to Cucumber Fermentation1 H. S. RAGHEB AND FR W. FABIAN Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan Received for publication December 28, 1956 The development of appreciable brine acidity in cu- cumber salt stock is necessary for curing the cucumbers and maintaining a desirable color during subsequent storage of stock. Several investigators (Le Fevre, 1920,1922; Joslyn, 1929; Fabian et al., 1932; Fellers et al., 1937; Fellers, 1939) recommended the addition of sugar to cucumber fermentations with the object of stimulat- ing the acid forming bacteria and producing more lactic acid. However, Fabian and Wickerham, (1935) showed that the addition of 1 per cent sucrose to barrels of genuine dills caused an initial increase of acid forming bacteria but not an increase in final acidity. Other studies by Jones et al. (1940) and Veldhuis et al. (1941) reported that the addition of sugar at the start of fer- mentation, or after 7 to 10 days, or in small amounts at short intervals throughout the fermentation, did not appreciably increase acid production even when the acid forming bacteria showed a significant rise in numbers. Lactobacillus plantarum is considered to be mainly responsible for acid production in cucumber fermen- tations (Etchells et al., 1946; Costilow et al., 1956), therefore, the presence of plentiful amounts of essential vitamins and amino acids for its growth is of great im- portance. Several workers (Rosen and Fabian, 1953; Costilow and Fabian, 1953a) have demonstrated that, during cucumber fermentation, the essential vitamins are present in sufficient amounts to satisfy the needs of L. plantarum. However, Costilow and Fabian (1953a, b) reported that great coliform and yeast activity caused a decrease in cystine and tryptophane levels respec- tively. They indicated that these two amino acids seem to be reduced during cucumber fermentation to a critical level for the growth of L. plantarum. Since corn steep liquor is an inexpensive industrial by-product, it could be used, if beneficial, on a com- mercial basis in cucumber fermentation. Little infor- mation, however, is available on its application for such a purpose. A patent by Pollack (1941) recom- mended the addition of one ounce corn steep liquor for every gallon of brine when making genuine dill pickles. The corn steep liquor treated barrels showed 1.4 per 1 Journal Articles Nos. 1876-77, Michigan Agrictultural Experiment Station. 253 cent lactic acid after 30 days, whereas, it was 0.6 per cent in control batches. Consequently, the object of this study was to inves- tigate the influence in cucumber fermentation of the addition of sugar, of cystine and tryptophane, and of corn steep liquor on the microbiological activity and acid formation by lactic acid bacteria. EXPERIMENTAL METHODS Semicommercial Fermentations In 1953 and 1954, 20 barrels were prepared outdoors at the salting station of the H. W. Madison Co., at Mason, Michigan. Cucumbers size 22 of the varieties MR-17 and SR-6 were used in 1953 and 1954 respec- tively. A 30° salometer schedule was followed in salt- ing. The different concentrations of sucrose, and cystine and tryptophane used in these experiments were de- termined on the basis of the weight of cucumbers and brine in each respective barrel. Corn steep liquor was added according to the volume of brine. The following treatments were carried out: (A) Sucrose treatments. Four barrels received 0.5 per cent sucrose which was added at the time of salting to 1 barrel and on the 7th day to the others. Each of 3 barrels was given 1.0 per cent sucrose. The sugar was added at the time of salting to the first barrel and on the 7th day to the second barrel. The third barrel re- ceived /% the amount of sugar at the time of salting, on the 7th day, and 13 on the 14th day. In this way, the influence of the addition of different concentrations of sucrose when added at different times during fermen- tation was investigated. (B) Cystine-tryptophane treatments. Each of 2 barrels received 0.0025 per cent cystine and 0.0025 per cent tryptophane which were added at the time of salting to 1 barrel and on the 7th day to the other. To investi- gate the effect of the combined treatment of the amino acids and sucrose, 3 barrels were prepared by adding 0.0025 per cent cystine, 0.0025 per cent tryptophane, and 1.0 per cent sucrose at the time of salting. Based on the studies of Costilow and Fabian (1953a) these con- 2 Size 2 cucumbers refers to cucumbers 3 to 4 in. long and 118 to 1% in. in diameter. on April 20, 2021 by guest http://aem.asm.org/ Downloaded from
Transcript
Addition of Sugar, Amino Acids and Corn Steep Liquor toCucumber Fermentation1
H. S. RAGHEB AND FR W. FABIAN
Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan
Received for publication December 28, 1956
The development of appreciable brine acidity in cu-cumber salt stock is necessary for curing the cucumbersand maintaining a desirable color during subsequentstorage of stock. Several investigators (Le Fevre,1920,1922; Joslyn, 1929; Fabian et al., 1932; Fellers et al.,1937; Fellers, 1939) recommended the addition of sugarto cucumber fermentations with the object of stimulat-ing the acid forming bacteria and producing more lacticacid. However, Fabian and Wickerham, (1935) showedthat the addition of 1 per cent sucrose to barrels ofgenuine dills caused an initial increase of acid formingbacteria but not an increase in final acidity. Otherstudies by Jones et al. (1940) and Veldhuis et al. (1941)reported that the addition of sugar at the start of fer-mentation, or after 7 to 10 days, or in small amountsat short intervals throughout the fermentation, didnot appreciably increase acid production even whenthe acid forming bacteria showed a significant rise innumbers.
Lactobacillus plantarum is considered to be mainlyresponsible for acid production in cucumber fermen-tations (Etchells et al., 1946; Costilow et al., 1956),therefore, the presence of plentiful amounts of essentialvitamins and amino acids for its growth is of great im-portance. Several workers (Rosen and Fabian, 1953;Costilow and Fabian, 1953a) have demonstrated that,during cucumber fermentation, the essential vitaminsare present in sufficient amounts to satisfy the needs ofL. plantarum. However, Costilow and Fabian (1953a, b)reported that great coliform and yeast activity causeda decrease in cystine and tryptophane levels respec-tively. They indicated that these two amino acidsseem to be reduced during cucumber fermentation toa critical level for the growth of L. plantarum.
Since corn steep liquor is an inexpensive industrialby-product, it could be used, if beneficial, on a com-mercial basis in cucumber fermentation. Little infor-mation, however, is available on its application forsuch a purpose. A patent by Pollack (1941) recom-mended the addition of one ounce corn steep liquor forevery gallon of brine when making genuine dill pickles.The corn steep liquor treated barrels showed 1.4 per
cent lactic acid after 30 days, whereas, it was 0.6 percent in control batches.
Consequently, the object of this study was to inves-tigate the influence in cucumber fermentation of theaddition of sugar, of cystine and tryptophane, and ofcorn steep liquor on the microbiological activity andacid formation by lactic acid bacteria.
EXPERIMENTAL METHODS
Semicommercial Fermentations
In 1953 and 1954, 20 barrels were prepared outdoorsat the salting station of the H. W. Madison Co., atMason, Michigan. Cucumbers size 22 of the varietiesMR-17 and SR-6 were used in 1953 and 1954 respec-tively. A 30° salometer schedule was followed in salt-ing. The different concentrations of sucrose, and cystineand tryptophane used in these experiments were de-termined on the basis of the weight of cucumbers andbrine in each respective barrel. Corn steep liquor wasadded according to the volume of brine. The followingtreatments were carried out:
(A) Sucrose treatments. Four barrels received 0.5 percent sucrose which was added at the time of salting to1 barrel and on the 7th day to the others. Each of 3barrels was given 1.0 per cent sucrose. The sugar wasadded at the time of salting to the first barrel and onthe 7th day to the second barrel. The third barrel re-ceived /% the amount of sugar at the time of salting,
on the 7th day, and 13 on the 14th day. In this way,the influence of the addition of different concentrationsof sucrose when added at different times during fermen-tation was investigated.
(B) Cystine-tryptophane treatments. Each of 2 barrelsreceived 0.0025 per cent cystine and 0.0025 per centtryptophane which were added at the time of saltingto 1 barrel and on the 7th day to the other. To investi-gate the effect of the combined treatment of the aminoacids and sucrose, 3 barrels were prepared by adding0.0025 per cent cystine, 0.0025 per cent tryptophane,and 1.0 per cent sucrose at the time of salting. Based onthe studies of Costilow and Fabian (1953a) these con-
2 Size 2 cucumbers refers to cucumbers 3 to 4 in. long and118 to 1% in. in diameter.
centrations of cystine and tryptophane were consideredmore than sufficient to provide the brine microorgan-isms with plentiful amounts of these amino acids.
(C) Corn steep liquor treatments. Two barrels receivedcorn steep liquor (1.0 oz for every gal of brine) whichwas added at the time of salting to 1 barrel and on the7th day to the other. The combined treatment of cornsteep liquor, in the same concentration, and 1.0 percent sucrose was studied by preparing 3 barrels. Thecorn steep liquor and sugar were divided into 3 equalportions and % added at the time of salting, % on the7th day, and 1 on the 14th day.
(D) No treatment. Three barrels were prepared fromthe same cucumbers at the same time as controls.
Method of SamplingA stainless steel tube 30 in. long and Yl6 in. in the
inside diameter with holes spaced 9.5 in. apart wasused for sampling. Samples were taken through a holemade in the head of each barrel by inserting the tubeand syphoning the brine into sterile sample tubes.During the first week, samples were taken every day.As the fermentation progressed, the periods of exami-nation were as indicated in the graphs. On removal ofthe brine samples from the barrels, they were immedi-ately transferred to the laboratory where chemical andbacteriological analyses were made, after which theywere frozen at - 18.5 C until the amino acid and vita-min assays were made.
Chemical and Bacteriological AnalysesThe following determinations were carried out:(1) Total titratable acidity: by titration with stand-
ard NaOH expressed as g of lactic acid per 100 ml ofbrine.
(2) Salt concentration: following the method ofKolthoff et al. (1929).
(3) Total sugar: by the Munson and Walker methodas outlined in Official Methods of Analysis of the Asso-ciation of Official Agricultural Chemist (1950). The re-sults were expressed as per cent invert sugar.
(4) pH of the brine: Cenco pH meter.(5) Total bacteria count: on dextrose tryptone agar
medium.(6) Lactic acid bacteria count: on V-8 medium de-
vised by Fabian et al. (1953).(7) Yeast count: on dextrose agar acidified with 5 ml
of 5 per cent tartaric acid per 100 ml of medium.(8) Coliform count: using single strength lauryl
tryptose broth inoculated in triplicate with variousdilutions of the brine samples.
All plates and tubes were incubated at 30 C for allmicrobial analyses. Three days' incubation were allowedfor the total and lactic acid bacteria counts; 2 days forcoliform and 7 days for yeast.
Amino Acid and Vitamin AssaysThe brine samples were removed from the freezer
and allowed to thaw at 4.5 C just prior to running theassays, then stored in the refrigerator under toluene.Assays for valine and glutamic acid were carried outfollowing the method of Sauberlich and Baumann(1946) using medium I. The dehydrated media pre-pared by Difco Laboratories, Inc., were used to assayfor biotin, niacin, pantothenic acid, valine, glutamicacid, and tryptophane. Streptococcus aquinus strain P-60(Leuconostoc mesenteroides) was used for the assays ofcystine, leucine, and isoleucine.
Laboratory Experiments under Ideal ConditionsAn experiment was designed to study sugar utiliza-
tion and acid production by L. plantarum under idealconditions. In this way it was hoped to show either theadvisability or futility of the addition of sugar; ofcystine and tryptophane; and of corn steep liquor tocucumber fermentation.For this purpose 32 flasks were prepared in the fol-
lowing manner:The cucumbers were washed thoroughly and the
juice was extracted. One hundred ml portions of thecucumber juice were placed in 500 ml Erlenmeyer flasks.To each flask 100 ml of 1.0 per cent NaCl was added,which made a final salt concentration of 0.5 per centafter which sucrose, cystine, and tryptophane, or cornsteep liquor, according to the specific treatment, wereadded. The flasks were sterilized at 15 lb for 15 min.Each flask was inoculated with 0.5 ml of an active pureculture of L. plantarum strain 17-5 suspended in physi-ological salt solution. All flasks were incubated at 30 C.The experiment covered a period of 35 days. Specifi-cally the following additions were made: 5 flasks, 0.5per cent sucrose; 5 flasks, 1.0 per cent sucrose; 4 flasks,0.0025 per cent cystine, 0.0025 per cent tryptophane; 4flasks, 0.0025 per cent cystine, 0.0025 per cent trypto-phane and 1.0 per cent sucrose; 4 flasks, 0.82 per centcorn steep liquor; 4 flasks, 0.82 per cent corn steepliquor and 1.0 per cent sucrose; and 6 flasks, no treat-ment (control). It should be noted that the concentra-tion of corn steep liquor used here is the same as thatused under semicommercial conditions. Samples weretaken periodically to determine the amount of acidformed and L. plantarum counts. Two flasks from eachspecific treatment were examined for the total sugar(expressed as invert sugar) in the brine at the startand at the end of the experiment.
RESULTS
Sugar Treatments(1) Effect of sugar on lactic acidformation. Before pre-
senting the effect of the different sugar treatments, a
study was made on the variations in lactic acid forma-tion that are normally present in brines of similar lots.Comparison between similar fermentations prepared in1953 and 1954 showed that the greatest difference inbrine acidity was 0.1 per cent. Therefore, it was as-sumed that, under semicommercial conditions, if aspecific treatment resulted in an increase greater than0.1 per cent lactic acid, the difference should be con-sidered significant.
In figure 1 are shown the average acidity values of allsugar treatments under semicommercial conditions.These results indicate no significant difference in brineacidity between the sugar and nonsugar treatments. Ingeneral, the amount of lactic acid increased from itslowest level at the time of salting to a maximum of 0.6per cent after fermentation for 15 to 20 days.
It should be mentioned here that the changes in pHlevels and salt concentrations were similar in both thesugar and nonsugar treatments.The laboratory experiments confirmed the previous
results. Figure 4 shows that acidity in both the sugarand nonsugar treatments was almost identical.
(2) Effect of sugar on total and lactic acid bacteriacounts. Comparison between similar fermentations pre-pared in 1953 and 1954 indicated that the largest dif-ference in lactic acid bacteria, expressed as log of count,was 1.4. Thus, it was safe to assume that differences of1.5 (32 times greater) or more in the log of count ofthese organisms are significant.Data compiled under semicommercial conditions
showed no indication of an appreciable increase in lacticacid bacteria numbers as a result of sugar addition.Only slight differences were observed. These small vari-ations, however, never exceeded the range of differencesthat might occur between individual barrels within onetreatment. The average lactic acid bacteria counts ofall sugar treatments under semicommercial conditions
0.7
a 0.60
o 0.5
0< 0.4
z 0.30wa. 0.2
0.1
5 10
are presented in figure 1. Their populations generallyincreased after salting and reached a peak within 5 to6 days. During the second week, the numbers remainedsomewhat high, but declined during the third week withvery low numbers observed after 41 days.The total bacteria numbers showed a similar trend
during fermentation. The counts resembled that oflactic acid formers with no significant increase in num-bers when sugar was added.
Determination of total sugar as invert sugar inbrines of the 1953 experiment was carried out to corre-late the changes in lactic acid bacteria populations withthe available sugar in the brine. Table 1 indicates thatin the control fermentation a rapid increase in thebrine sugar contents occurred after salting. This wasmostly due to leaching of the naturally occurringsugars from the cucumbers into the brine. The totalsugar content, expressed as invert sugar, reached amaximum about the 7th day and could not be detectedon the 25th day. This decline was most likely due tothe utilization of sugar by the brine microorganisms.In the barrels where sucrose was added, greater amountsof invert sugar were detected in the brine at the startof fermentation. However, the levels of invert sugar de-creased to a level comparable to that of the control in-dicating greater utilization of sugars by the brinemicroorganisms. Yet, as is shown in figure 1, theamounts of lactic acid formed were similar to that ofthe control. Apparently then, the added sucrose wasmainly utilized by organisms other than lactic acidbacteria.Under laboratory conditions, no significant differ-
ences in L. plantarum counts were observed betweenthe sugar and nonsugar treatments. Furthermore, theamounts of sugar utilized by L. plantarum were similar(table 2). The added sucrose was not converted intolactic acid. This would indicate that L. plantarum can
15 20 25 30 35 40 45TIME IN DAYS
10.0
9.0 a
w8.0 al
1--z
70 :0
6.0 0
05.0 °
4.0
3.0
FIG. 1. Influence of addition of sugar in brines of semicommercial cucumber fermentations on lactic acid and acid formingbacteria.
000 - o - -
460 ~~~~0A-f AIQsS Acidity Acid-formers
4Io ._- ._- Control (avg.of 3)A O --o A--6 Sugar treatments
use only a certain amount of sugar and produce a cer-tain amount of acid irrespective of the amount of sugarpresent in the brine.
(3) Effect of sugar on yeast and coliform populations.Comparison between similar barrels prepared in 1953and 1954 showed that a variation of 2.1 in the log ofyeast count might normally occur. Accordingly, a dif-ference greater than 2.1 (approximately 100-fold) wasconsidered significant.
In figure 2, part B, it is shown that addition of sugar
j
zD00IL000-J 7.0 *- Control (avg. of 3)
. O_ _0 Sugar treatments at timeof salting (avg. of 2)
6.0-
4.0 ^= l| = ^ Ze _t j A e f 0
2.0.. -_ 0 5 10 15 20 25 30 35 40
TIME IN DAYS45 50
FIG. 2. Influence of addition of sugar to brines of semi-commercial cucumber fermentation on yeasts.
0
40
04-J
I--zIJ
cr.w0.
at the time of salting caused no significant increasein yeast population over the control. However, when 0.5or 1.0 per cent was added on the 7th day, the yeastcounts immediately increased (figure 2, part A). Thelog of yeast count always exceeded 2.1. The numbersthen rapidly declined until they reached a level similarto that of the control. When sugar was divided into threeequal portions and added at different times throughoutthe fermentation, only the second addition of sugar onthe 7th day resulted in a significant rise in yeast popu-lation over the control (figure 2, part A).
In contrast to the lactic acid bacteria, yeasts, whichhad generally decreased in numbers during the first fewdays after salting, started rapid growth after about 7days and reached their maximum population in from 10to 15 days.As to coliforms, they disappeared from all barrels after
5 days. It is believed that they played no significant partin these fermentations.
Cystine and Tryptophane Treatments
Effect of added cystine and tryptophane on lactic acidproduction. Since a variation greater than 0.1 per centacid between different treatments was previously con-sidered significant, this variation value was also usedhere as basis for comparison between the specific treat-ments and the control.
In all barrels receiving cystine and tryptophane,alone or in combination with sucrose, brine acidity wasnot appreciably higher than the control. The averageacidity values of all cystine and tryptophane treatmentsare graphically presented in figure 3. Since no greateramounts of acid were formed, the addition of cystineand tryptophane, under the conditions of these ex-periments, was of no value in increasing lactic acid pro-duction.
In table 1 it is shown that the utilization of sugar bythe brine microorganisms was similar to that of the con-
-j
wa.
z
30
UIL.0CD0-i
TIME IN DAYS
FIG. 3. Influence of addition of cystine and tryptophane to brines of semicommercial cucumber fermentations on lactic acid andacid forming bacteria.
trol. Since brine acidity was not appreciably affected,
the cystine-tryptophane treatments did not stimulatethe conversion of the naturally occurring sugars in thecucumbers to lactic acid.The previous results were confirmed under laboratory
1.40-
1.20
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< 1.40
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L. plontorumcounts Acid ity
* Control (avg. of 4)o---o Sugar treatments
(avg. of 6)
A
*-C Control (ovg. of 4)0---o Cystine a tryptophone
treatments (avg. of 4)
B
I
.
<.V j-i
1.0 zXCL
9.0
7.0 00
U.
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M% j0.60 ~~~~~~~~~~~~3.0J*-A 0*~ --- Control (avg. of 4)
FIG. 4. Influence of addition of sugar, addition of cystineand tryptophane, and addition of corn steep liquor to brinesof laboratory cucumber fermentations on lactic acid andLactobacillus plantarum.
conditions; the cystine-tryptophane treatments showedno effect on the total and lactic acid bacteria counts as
well as yeasts. Figures 3 and 4 present the average
lactic acid bacteria populations of all cystine andtryptophane treatments under semicommercial andlaboratory conditions respectively. Figure 5, part B,shows the average yeast numbers.The coliform populations varied considerably from
one barrel to another. There was no indication that thecystine tryptophane treatments had any significanteffect on the number of coliforms during fermentation.In general, coliforms disappeared from all fermenta-tions after 6 days. Apparently, unfavorable conditionsin the brine inhibited them.
Corn Steep Liquor Treatments
(1) Effect of added corn steep liquor on lactic acid pro-
duction. When corn steep liquor was added to the bar-rels, brine acidity was similar to that of the controlduring the first 2 weeks of fermentation. Thereafter,acidity was slightly higher. Although there is still thepossibility that variations of such magnitude mightoccur between duplicate fermentations, yet, in all bar-rels receiving corn steep liquor, lactic acid productionwas considerably higher than the control. Furthermore,this slight increase of acidity lasted for a considerableperiod of time. Figure 6 presents the average acidityvalues of all corn steep liquor treatments examined.The invert sugar contents of the different brines and
consequently the total sugar levels showed a rapid in-crease after salting. After reaching a maximum, thelevels decreased at a more rapid rate than in the con-
trol. For example, when corn steep liquor was added atthe time of salting, or on the 7th day, no invert sugarwas detected on the 9th day as compared to 0.7 per
cent for the control (table 1). Also, when corn steep
TABLE 1. Per cent total sugar* in brines at semicommercial cucumber fermentations
treatment Sucrose at Sucrose on Sucrose at Sucrose on Sucrose acids at acids on acids an liquor at liquor on suor(control) salting 7th day salting 7th day (divided) salting 7th day salting salting 7th day (divided
0-3 hr t 1.254 t 1.191 t 0.673 t t t t t 0.6872 days 0.331 1.269 0.395 1.730 0.436 0.820 0.318 0.318 1.862 0.450 0.423 0.7826 days 1.008 1.697 1.013 1.964 0.882 1.234 0.963 0.927 2.000 0.906 1.037 1.1328 days 0.821 1.329 1.637 1.810 2.144 1.359 0.621 0.714 1.835 0.327 0.668 0.7329 days 0.777 1.003 1.029 1.529 2.024 0.791 0.593 0.782 1.316 0.327 0.165 0.418
11 days 0.725 0.615 0.512 0.968 0.646 0.418 0.502 0.427 0.712 1 t 0.26813 days 0.336 0.386 0.469 0.624 _ 0.422 0.361 0.295 0.515 -15 days 0.091 0.407 0.317 0.055 0.109 - 0.30525 days 0.151 0.114 0.241 0.250 0.13633 days 0.165 0.138 0.173 0.290 t t 0.14741 days 4 0.056 0.062 0.080 0.069 t 1 0.122 t
* Total sugar expressed as grams invert sugar per 100 ml of brine.t Average of two determinations.t Less than 0.045 g invert sugar per 100 ml of brine.
liquor was added in combination with 1.0 per centsucrose, the invert sugar levels generally decreasedduring the fermentation indicating its rapid utilizationby the brine microorganisms. However, the brineacidity was similar but not greater than where onlycorn steep liquor was added without sugar. Therefore,the added sucrose was not converted to lactic acid. Onecan conclude then that the slight increase in brineacidity observed over the control in all corn steepliquor treatments was probably due to greater utiliza-
TABLE 2. Per cent total sugar* in brines of laboratory cucumberfermentations
Per Cent Invert Sugart
Treatments
Time No Amino Corntreat- 0.5% 1.%Amn acids Cor steepment Su u cd and liquorancon- crose crose cs u1- liquor aSntrol crose crose
O hr 1. 284 1.926 2.230 1.294 2.434 1.334 2.25235 days 0. 381 0.944 1.358 0.341 1.379 t 0.899
* Average of two determinations.t Total sugar expressed as grams invert sugar per 100 ml of
brine.t Less than 0.045 g invert sugar per 100 ml of brine. It was
considered equal to 0.045 g when the amount of sugar utilizedwas calculated.
-i
cr.w
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0
L.
0
0
-i
B *- - Control (avg. of 3)7.0 - o-.-o Cystine a tryptophone treatments
(avg. of 3)6.0
50t <@~~5.0
4.0 00,
3.0 *0
2.0 p20o 5 10 15 20 25 30 35 40 45 50
TIME IN DAYS
FIG. 5. Influence of addition of cystine and tryptophane, andaddition of corn steep liquor to brines of semicommercialcucumber fermentations on yeasts.
tion of the carbohydrates in the cucumbers by lacticacid bacteria.The laboratory experiments agreed with the previous
results. Figure 4 shows addition of corn steep liquor,alone or in combination with 1.0 per cent sucrose, re-sulted in high acidity levels on the 11th day and there-after. This increase in acidity is significant since it ex-ceeded by far the variations found between duplicatedeterminations.The total sugar contents showed that L. plantarum
was able to utilize more of the naturally occurring sugarin the cucumbers when corn steep liquor was added tothe flasks. Table 2 indicates that the amount of invertsugar utilized was 1.3 per cent in the corn steep liquortreated flasks, whereas, only 0.9 per cent was observedin the control. The combined treatment of corn steepliquor and sucrose showed almost the same results.Thus, the added sucrose was not utilized by L.plantarum even in the presence of corn steep liquor.
(2) Effect of added corn steep liquor on microbiologicactivity. The average populations of lactic acid bacteriain 5 corn steep liquor treated barrels and 3 controls isillustrated in figure 6. It is evident from this graph thatthere was no significant difference during the first 2weeks of fermentation between the corn steep liquortreatments and the control. However, their numberswere higher than those of the control on the 29th dayand thereafter. The log of count of these organisms washigher than the control by at least 1.7 (about 50 timesgreater).The over-all total bacterial changes during fermenta-
tion resembled those of the acid formers.Under laboratory conditions, L. plantarum counts
were higher than the control about the 15th day andthereafter (figure 4).
Population changes of yeasts during fermentation of5 barrels of cucumbers supplemented with corn steepliquor are shown in figure 5, part A. In general, yeastsreached a peak during the second week of fermentationbut rapidly declined to a level lower than the controlabout the 18th day and maintained lower levels duringthe rest of the examination period. There was someevidence that combining sucrose with corn steep liquormight have some effect on yeast population. Their num-bers significantly increased when the treatment wasmade on the 7th day.As to coliforms, the corn steep liquor treatments had
no effect on their numbers during fermentation.
Availability of Vitamins and Amino AcidsIn general, the vitamins; biotin, niacin, pantothenic
acid; and the amino acids; leucine, isoleucine, cystine,valine, and glutamic acid gradually increased afterbrining. This was mostly due to withdrawing of thenutrients from the cucumbers by osmosis. After reach-
ing a maximum, they maintained high levels to the endof the examination period.Tryptophane was the only amino acid affected
by fermentation. Its levels reached a peak in from 6to 10 days, then markedly declined. This trend ofchanges was observed in all sugar and cystine-trypto-phane treatments (figures 7 and 8). A good illustrationof the tryptophane decline occurred when cystine andtryptophane were added on the 7th day (figure 8).Since no greater amounts of acid were formed as a re-sult of these treatments, tryptophane might have beena limiting factor. However, it was noted that thepresence of plentiful amounts of cystine and trypto-phane during the active fermentation period was of novalue in enhancing brine acidity.When corn steep liquor was added alone, tryptophane
decreased slightly at the end of the examination periodas compared with a large decline in case of the control(figure 9). Evidently, smaller amounts of tryptophanewere utilized by the brine microorganisms. This corre-lates with the results that lower yeast populations werefound after 18 days. About the same time, lactic acidbacteria maintained somewhat higher numbers thanin the control. It has been previously reported thatgreater utilization of sugar was observed when cornsteep liquor was added. Apparently then, the addedcorn steep liquor enhanced the utilization of the avail-able sugar in the brine resulting in the production ofgreater amounts of lactic acid. When the combinedtreatment of corn steep liquor and sucrose was applied,the decline in tryptophane levels was greater at the endof the examination period than in the cases where cornsteep liquor was added alone. However, a similar in-crease in acidity occurred. This would indicate thattryptophane was mostly utilized by yeasts and not bylactic acid bacteria.
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DISCUSSION
In this investigation, addition of 0.5 or 1.0 per centsucrose did not accelerate acid formation. The increasedacid production reported by many workers was probablyonly slight variations that normally occur between in-dividual fermentations. Total and lactic acid bacterialpopulations were not appreciably affected by the differ-ent sugar treatments. Normal variations in bacterialpopulations are probably responsible for discrepanciesin the results of other studies.Under presumably optimum conditions, the utiliza-
tion of total sugar was not enhanced by increasing thesugar concentration in the brine. As a matter of fact,L. plantarum strain 17-5 was not able to utilize all thenaturally occurring carbohydrates in the cucumbers.The carbohydrate level available in the brine was nota limiting factor for the growth of these organisms.No appreciable effect on yeast numbers was observed
when sugar was added at the time of salting. Ap-parently yeasts, which were introduced into the barrelsby adhering to the cucumbers, were becoming adjustedto environmental conditions. As fermentation pro-gressed, their adjustment was most likely the influenc-ing factor on their numbers. The rapid increase in yeastpopulation which was noted when sugar was added onthe 7th day was probably due to the presence of a richsource of carbohydrates which they immediately uti-lized. When the sugar in the brine was considerablylowered, a corresponding decline in yeast numbersoccurred.
Addition of cystine and tryptophane showed noeffect on total titratable acidity and microbiologic ac-tivity of cucumber fermentations.
Addition of corn steep liquor to the cucumber saltstock barrels enhanced the production of lactic acidduring the third week of fermentation and thereafter.
_-5_ 0 10.0
- _ 9.0 i
ity Acid-formers 8.0
-- A-A Control (avg.of 3)-0 Corn steep liquor 7.0 Z
treatments (avg. of 5) o60 E
05.0
5 10 15 20 25 30 35 40 45TIME IN DAYS
FIG. 6. Influence of addition of corn steep liquor to brines of semicommercial cucumber fermentations on lactic acid and acidforming bacteria.
During the same period, high population of lactic acidbacteria were present, whereas, yeast numbers werelow.With the exception of tryptophane, studies on the
vitamins and amino acids which are essential for the
j *v.v
0: 5.0a.
o
Cz4a.
° 30.0a.1- 25.0
:A20.0115.0
10.0
5.0
0 5 10 15 20 25 30 35 40 45TIME IN DAYS
FIG. 7. Influence of addition of sugar to brines of semicommerical cucumber fermentations on tryptophane.
O O Cystine a tryptophone50.0 at salting
i^___ Cystine a tryptophoneon 7th day
X 450 Cystins, tryptophone akL 400 j\ + sugar at solting
(L 40.0-
w A43 5.0 -
!
x
oJ0
2050\04IL~~~
15.0P
10.Oj
5.0
5 10 IS5 20 25 30 35 40 45 50
TIME IN DAYS
FIG. 8. Influence of addition of cystine and tryptophaneto brines of semicommercial cucumber fermentations on
tryptophane.
growth of L. plantarum indicated than an abundantsupply of these nutrients was present at all times duringfermentation. However, Costilow and Fabian (1953a)reported that cystine might possibly be reduced to acritical level under certain conditions. Since Aerobacterfermentation in this study was insignificant, no sucheffect on the cystine levels was observed.
In all sugar and cystine-tryptophane treatments,tryptophane levels declined after the second week offermentation. Thus, it seemed at first that tryptophanemight have an important role in acid production.However, addition of corn steep liquor in combinationwith sugar stimulated acid formation, whereas, tryp-tophane levels were greatly lowered during the fermen-tation. Therefore, under the conditions of these experi-ments, tryptophane was not limiting for the productionof lactic acid. Apparently, greater yeast activity wasresponsible for this reduction. Furthermore, in allbarrels examined, tryptophane levels declined whenyeast activity was greatest. It was probably utilized ordestroyed by yeasts. These results agree with those ofRosen and Fabian (1953) and Costilow and Fabian(1953a, b).
Results have also demonstrated that in all corn steepliquor treatments, there was a rapid utilization of sugarby the brine microorganisms. Thus, corn steep liquormight contain a substance or substances, other thanthose examined, which enhanced the utilization of thenaturally occurring sugars in the cucumbers.Another possibility might be that corn steep liquor
provides an unknown factor (Heimbuch et al., 1956)when its concentration declines in the brine to a criticallevel. Several workers have shown that corn steep liquor
20 25 30 35 40 45TIME IN DAYS
FIG. 9. Influence of addition of corn steep liquor to brinesof semicommercial cucumber fermentations on tryptophane.
is a good source of soluble protein derivatives and min-erals as well as growth promoting substances (Bowdenand Peterson, 1946; Stefaniak et al., 1946). Sincearginine, alanine, phenylalanine, and tyrosine havebeen shown sometimes to be stimulatory or essentialfor L. plantarum (Lyman et al., 1947), further investiga-tions along these lines would be of great interest.
SUMMARYStudies were made during two seasons under semi-
commercial and laboratory conditions of the influenceof the addition of sugar, the addition of cystine andtryptophane, and the addition of corn steep liquor on
the production of lactic acid, the microbiologic activity,and the availability of certain vitamins and amino acidsin cucumber fermentation.Under the conditions of these experiments, acid pro-
duction was not stimulated by the addition of sugar or
cystine and tryptophane. Corn steep liquor, however,enhanced acid formation. It also increased the utiliza-tion of sugar in the brine.Under ideal conditions, Lactobacillus plantarum
strain 17-5 was capable of producing only a certainamount of lactic acid irrespective of the amount of sugaror cystine and tryptophane in the brine.No lasting effect was observed on the total and lactic
acid bacteria populations in all sugar and cystine-tryptophane treatments. Addition of corn steep liquor,however, stimulated the bacterial flora.Only the addition of sugar on the 7th day of fermenta-
tion caused an immediate increase in yeast populationwhich was followed by a rapid decline when the totalsugar in the brine was considerably lowered.Tryptophane levels consistently decreased when yeast
activity was greatest.The possibility that corn steep liquor might contain a
substance or substances, other than those examined,which stimulated lactic acid production is indicated.
REFERENCES
BOWDEN, J. P. AND PETERSON, W. H. 1946 The role of corn
steep liquor in the production of penicillin. Arch. Bio-chem., 9, 387.
COSTILOW, R. N., COUGHLIN, F. M., ROBACH, D. L., AND
RAGHEB, H. S. 1956 A study of the acid-forming bac-teria from cucumber fermentations in Michigan. FoodResearch, 21, 27-33.
COSTILOW, R. N. AND FABIAN, F. W. 1953a Availability ofessential vitamins and amino acids for Lactobacillusplantarum in cucumber fermentations. Appl. Microbiol.,1, 320-326.
COSTILOW, R. N. AND FABIAN, F. W. 1953b Effect of variousmicroorganisms on the vitamin and amino acid content ofcucumber brines. Appl. Microbiol., 1, 327-329.
ETCHELLS, J. L. AND JONES, I. D. 1946 Characteristics oflactic acid bacteria from commerical cucumber fermenta-tions. J. Bacteriol., 52, 593-599.
FABIAN, F. W., BRYAN, C. S., AND ETCHELLS, J. L. 1932 Ex-perimental work in cucumber fermentation. MichiganAgr. Expt. Sta., Tech Bull., 126.
FABIAN, F. W., FULDE, R. C., AND MERRICK, J. E. 1953 Anew medium for determining lactobacilli. Food Re-search, 18, 280-289.
FABIAN, F. W. AND WICKERHAM, L. J. 1935 -Experimentalwork on cucumber fermentation. Michigan Agr. Expt.Sta., Tech Bull., 146.
FELLERS, C. R. 1939 Experiments with dextrose in picklesand kraut. Fruit Products J., 18, 238.
FELLERS, C. R., MILLER, J., AND ONADORF, T. 1937 Dex-trose in the manufacturing of fruit and vegetable prod-ucts. Ind. Eng. Chem., 29, 946-949.
HEIMBUCH, A. H., AURAND, L. W., AND SPECK, M. I. 1956Some characteristics of a growth stimulant in corn steepliquor for Lactobacillus casei. J. Bacteriol., 72, 543-547.
JONES, I. D., VELDHUIS, M. K., ETCHELLS, J. L., AND VEER-HOFF, 0. 1940 Chemical and bacteriological changes indill pickle brines during fermentation. Food Research, 5,533-547.
JOSLYN, M. A. 1929 Some observations on the softening ofdill pickles. Fruit Products J., 8 (No. 8), 19-21 (No. 9),16-17.
KOLTHOFF, I. M., LAUER, W. M., AND LUNDS, C. J. 1929 Theuse of dichlorofluorescein as an absorption indicator forthe argentometric titration of chlorides. J. Am. Chem.Soc., 51, 3273.
LEFEvRE, E. 1920 Pickle processing investigations. Can-ner, 50, 230-232.
LEFEvRE, E. 1922 Bacteriology of sauerkraut and pickles.Chem. Age (London), 30, 24.
LYMAN, C. M., MOSELEY, O., WOOD, S., BUTLER, B., AND HALE,P. 1947 Some chemical factors which influence theamino acid requirements of lactic acid bacteria. J. Biol.Chem., 167, 177-187.
Official Methods of Analysis of the Association of Official Agri-cultural Chemist 1950 7th ed. Washington, D. C.
POLLAK, A. 1941 Process of pickling, curing, and preservingfruits and vegetables. U. S. Patent No. 2,322,880.
ROSEN, S. AND FABIAN, F. W. 1953 The importance of biotin,niacin, and pantothenic acid in cucumber fermentation.Food Technol., 7, 244-247.
STEFANIAK, J. J., GAILEY, F. B., BROWN, C. S., AND JOHNSON,M. J. 1946 Pilot plant equipment for submerged pro-duction of penicillin. Ind. Eng. Chem., 38, 666.
SAUBERLICH, H. E. AND BAUMANN, C. A. 1946 The effect ofdietary protein upon amino acid excretion by rats andmice. J. Biol. Chem., 166, 417-428.
VELDHUIS, M. K., ETCHELLS, J. L., JONES, I. D., AND VEER-HOFF, 0. 1941 Influence of sugar additions to brines inpickle fermentation. Food Inds., 13, (No. 10), 54-56, (No.11), 48-50.
